Heng Wang

Homozygosity for a novel splice site mutation in the cardiac myosin-binding protein C gene causes severe neonatal hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is typically inherited in an autosomal dominant pattern and has a variable age of onset and prognosis. Mutations in the myosin‐binding protein C (MYBPC3) gene are one of the most frequent genetic causes of the disease. Patients with MYBPC3 mutations generally have a late onset and a relatively good prognosis. We report here more than 20 Old Order Amish children with severe neonatal hypertrophic cardiomyopathy caused by a novel homozygous splice site mutation in the MYBPC3 gene. The affected children typically presented with signs and symptoms of congestive heart failure during the first 3 weeks of life. Echocardiography revealed hypertrophic non‐obstructive cardiomyopathy. These children had a life span averaging 3–4 months. All patients died from heart failure before 1 year of age unless they received a heart transplant. A genome‐wide mapping study was performed in three patients. The disease related gene was localized to a 4.6 Mb region on chromosome 11p11.2‐p11.12. This homozygous block contained MYBPC3, a previously identified cardiomyopathy related gene. We identified a novel homozygous mutation, c.3330u2009+u20092Tu2009>u2009G, in the splice‐donor site of MYBPC3 intron 30. The mutation resulted in skipping of the 140‐bp exon 30, which led to a frame shift and premature stop codon in exon 31 (p.Asp1064GlyfsX38). We have found a substantial incidence of this phenotype in Old Order Amish communities. It is also concerning that many unidentified heterozygous individuals who are at risk for development of hypertrophic cardiomyopathy do not receive proper medical attention in the communities.

A homozygous missense mutation in HERC2 associated with global developmental delay and autism spectrum disorder

We studied a unique phenotype of cognitive delay, autistic behavior, and gait instability segregating in three separate sibships. We initiated genome‐wide mapping in two sibships using Affymetrix 10K SNP Mapping Arrays and identified a homozygous 8.2 Mb region on chromosome 15 common to five affected children. We used exome sequencing of two affected children to assess coding sequence variants within the mapped interval. Four novel homozygous exome variants were shared between the two patients; however, only two variants localized to the mapped interval on chromosome 15. A third sibship in an Ohio Amish deme narrowed the mapped interval to 2.6 Mb and excluded one of the two novel homozygous exome variants. The remaining variant, a missense change in HERC2 (c.1781C>T, p.Pro594Leu), occurs in a highly conserved proline residue within an RCC1‐like functional domain. Functional studies of truncated HERC2 in adherent retinal pigment epithelium cells suggest that the p.Pro594Leu variant induces protein aggregation and leads to decreased HERC2 abundance. The phenotypic correlation with the mouse Herc1 and Herc2 mutants as well as the phenotypic overlap with Angelman syndrome provide further evidence that pathogenic changes in HERC2 are associated with nonsyndromic intellectual disability, autism, and gait disturbance. Hum Mutat 33:1639–1646, 2012.

Homozygous frameshift mutation in TMCO1 causes a syndrome with craniofacial dysmorphism, skeletal anomalies, and mental retardation

We identified an autosomal recessive condition in 11 individuals in the Old Order Amish of northeastern Ohio. The syndrome was characterized by distinctive craniofacial dysmorphism, skeletal anomalies, and mental retardation. The typical craniofacial dysmorphism included brachycephaly, highly arched bushy eyebrows, synophrys, long eyelashes, low-set ears, microdontism of primary teeth, and generalized gingival hyperplasia, whereas Sprengel deformity of scapula, fusion of spine, rib abnormities, pectus excavatum, and pes planus represented skeletal anomalies. The genome-wide homozygosity mapping using six affected individuals localized the disease gene to a 3.3-Mb region on chromosome 1q23.3-q24.1. Candidate gene sequencing identified a homozygous frameshift mutation, c.139_140delAG, in the transmembrane and coiled-coil domains 1 (TMCO1) gene, as the pathogenic change in all affected members of the extended pedigree. This mutation is predicted to result in a severely truncated protein (p.Ser47Ter) of only one-fourth the original length. The TMCO1 gene product is a member of DUF841 superfamily of several eukaryotic proteins with unknown function. The gene has highly conserved amino acid sequence and is universally expressed in all human tissues examined. The high degree of conservation and the ubiquitous expression pattern in human adult and fetal tissues suggest a critical role for TMCO1. This report shows a TMCO1 sequence variant being associated with a genetic disorder in human. We propose “TMCO1 defect syndrome” as the name of this condition.

A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-β glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.

A nonsense mutation of PEPD in four amish children with prolidase deficiency

Encoded by the peptidase D (PEPD) gene located at 19q12‐q13.11, prolidase is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of oligopeptides with a C‐terminal proline or hydroxyproline. We describe here four Amish children with a severe phenotype of prolidase deficiency in the Geauga settlements of Ohio as the first report of prolidase deficiency in the Amish population as well as in the United States. The patients presented with infection, hepatosplenomegaly, or thrombocytopenia, in contrast to most cases previously reported in the literature, presenting with skin ulcers. All four patients had typical facial features, classic skin ulcers, and multisystem involvement. Recurrent infections, asthma‐like chronic reactive airway disease, hyperimmunoglobulins, hepatosplenomegaly with mildly elevated aspartate transaminase (AST), anemia, and thrombocytopenia were common and massive imidodipeptiduria was universal. Prolidase activity in our patients is nearly undetectable. Direct sequencing of PCR‐amplified genomic DNA for all of the exons from the four patients revealed the same homozygous single nucleotide mutation c.793 Tu2009>u2009C in exon 11, resulting in a premature stop‐codon at amino acid residue 265 (p.R265X). It is speculated that the severe phenotype in these patients might be associated with the type of the PEPD gene mutation.

Prolidase deficiency breaks tolerance to lupus‐associated antigens

Prolidase deficiency is a rare autosomal recessive disease in which one of the last steps of collagen metabolism, cleavage of proline‐containing dipeptides, is impaired. Only about 93 patients have been reported with about 10% also having systemic lupus erythematosus (SLE).

Phenylalanine hydroxylase deficiency exhibits mutation heterogeneity in two large old order Amish settlements.

Phenyalanine hydroxylase (PAH) deficiency (OMIM 261600) results in intolerance to dietary phenylalanine and produces a spectrum of disorders including phenylketonuria (PKU) and non-PKU hyperphenylalaninemia. PAH deficiency, the most common inborn error of amino acid metabolism in Caucasians with an incidence of about 1/10,000, is inherited in an autosomal recessive manner. According to PAHdb, a public, on-line, locus-specific mutation database (http://www.pahdb.mcgill.ca/), over 500 causative mutations have been identified in this phenotypically heterogeneous metabolic disorder. Historically, identification of pathogenic PAH alleles in different populations has provided unique opportunities to study important issues related to human population genetics, such as migration, genetic drift, and founder effects [Scriver and Kaufman, 2000]. The Old Order Amish are a religious sect that emigrated from Europe in the mid-1700s. Individual Amish settlements throughout North America have remained isolated through social and religious mechanisms. The founder effect in such isolated populations often appears pronounced, as demonstrated by the high incidence of some otherwise rare genetic disorders [McKusick, 1978; Morton et al., 2003]. Here, we report on PAH mutation analysis in PKU patients from two large Old Order Amish settlements in the United States; the Lancaster County, PA, and Geauga County, OH, settlements. Although the twopopulations are socially similar and share many common founders, this study demonstrated unexpected mutation heterogeneity between and within the two populations. The PKU patients included in this study were previously identified through state newborn screening programs; the diagnosis was confirmed by plasma amino acid profiles. Affected individuals, as well as parents and siblings, were recruited to participate in the study and informed consent was obtained from each participant. DNA was isolated from peripheral blood with commercially available DNA purification kits. The 13 coding exons of the PAH gene were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers and subsequently sequenced on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Multiple patients from the Lancaster County settlement and the index case from the Geauga County settlement were subject to complete PAH gene sequencing. Genotyping in additional patients and their families concentrated on the populationspecific mutations identified in each community. Genealogical data obtained from participating families was confirmed and expanded based on private or published family records provided by the local Amish communities. The Geauga County Amish settlement was founded in 1886, with initial immigrants from HolmesCounty,OH. Later, Amish fromPennsylvania settlements joined them, creating the extant population in Geauga County. In this settlement,14 individualswith classic PKUhavebeen identified from four families. The closest common ancestral couple (JM (1817–1890) and LT (1819–1865)) was seven or eight generations removed from all patients (Fig. 1).

Oral Ganglioside Supplement Improves Growth and Development in Patients with Ganglioside GM3 Synthase Deficiency

Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GM3D) causes an absence of GM3 and all downstream biosynthetic derivatives. The affected individuals manifest with severe irritability, intractable seizures, and profound intellectual disability. The current study is to assess the effects of an oral ganglioside supplement to patients with GM3D, particularly on their growth and development during early childhood. A total of 13 young children, 11 of them under 40xa0months old, received oral ganglioside supplement through a dairy product enriched in gangliosides, for an average of 34xa0months. Clinical improvements were observed in most children soon after the supplement was initiated. Significantly improved growth and development were documented in these subjects as average percentiles for weight, height, and occipitofrontal circumference increased in 1-2xa0months. Three children with initial microcephaly demonstrated significant catch-up head growth and became normocephalic. We also illustrated brief improvements in developmental and cognitive scores, particularly in communication and socialization domains through Vineland-II. However, all improvements seemed transient and gradually phased out after 12xa0months of supplementation. Gangliosides GM1 and GM3, although measureable in plasma during the study, were not significantly changed with ganglioside supplementation for up to 30xa0months. We speculate that the downstream metabolism of ganglioside biosynthesis is fairly active and the potential need for gangliosides in the human body is likely substantial. As we search for new effective therapies for GM3D, approaches to reestablish endogenous ganglioside supplies in the affected individuals should be considered.